Abstract：We evaluated the role of Dmrt1 in regulating sex determination and differentiation in half-smooth tongue sole (Cynoglossus semilaevis). The recombinant protein of C. semilaevis Dmrt1 was expressed by the prokaryotic expression system (Pet-32a) and the protein (Pet-32-dmrt1) was subsequently purified by immobilized-metal affinity chromatography. We measured gene expression after injection of the recombinant protein Pet-32-Dmrt1 into the ovary. The expression of CYP19A and Foxl2 decreased significantly between 6 and 24 h after injection and the mRNA transcription level of Sox9aa was significantly increased between 6 and 24 h. However, the expression of all three genes expression returned to normal 48 h after injection. Our results indicate that Dmrt1 recombinant protein has biological activity, and plays a role in sex differentiation by regulating sex related gene expression.